Evidence of abnormality in glutathione metabolism in the airways of preterm born children with a history of bronchopulmonary dysplasia

Preterm-born children are at risk of long-term pulmonary deficits, including those who developed bronchopulmonary dysplasia (BPD) in infancy, however the underlying mechanisms remain poorly understood. We characterised the exhaled breath condensate (EBC) metabolome from preterm-born children, both with and without BPD. Following spirometry, EBC from children aged 7–12 years, from the Respiratory Health Outcomes in Neonates study, were analysed using Time-of-Flight Mass Spectrometry. Metabolite Set Enrichment Analysis (MSEA) linked significantly altered metabolites to biological processes. Linear regression models examined relationships between metabolites of interest and participant demographics. EBC was analysed from 214 children, 144 were born preterm, including 34 with BPD. 235 metabolites were detected, with 38 above the detection limit in every sample. Alanine and pyroglutamic acid were significantly reduced in the BPD group when compared to preterm controls. MSEA demonstrated a reduction in glutathione metabolism. Reduced quantities of alanine, ornithine and urea in the BPD group were linked with alteration of the urea cycle. Linear regression revealed significant associations with BPD when other characteristics were considered, but not with current lung function parameters. In this exploratory study of the airway metabolome, preterm-born children with a history of BPD had changes consistent with reduced antioxidant mechanisms suggesting oxidative stress.

Bronchopulmonary dysplasia (BPD), also known as chronic lung disease of prematurity, is the one of the commonest respiratory consequences of preterm birth and, despite advances in neonatal care over the last twenty years, rates of BPD are continuing to rise 1 .The pathogenesis of BPD is multifactorial, but pulmonary injury secondary to oxygen free radical production and inflammation form an important common pathway leading to altered lung development 2,3 .A history of preterm birth, both with and without a history of BPD, has been consistently associated with poorer lung function in later life 4 , and there is growing evidence that those with a history of BPD risk the development of chronic obstructive pulmonary disease (COPD) in early adulthood 5,6 , as well as being diagnosed with asthma 7 , although there is increasing recognition that prematurityassociated lung disease (PLD) has different underlying mechanism to asthma 8 .Lung function continues to develop throughout childhood and adolescence, with increasing number of alveoli, airway size and lung volume 9 , before declining after early adulthood 10 .Preterm-born individuals have been shown to have significantly lower forced expiratory volume in 1 s (FEV 1 ) than those born at term 11 , therefore providing a potential therapeutic window of opportunity for optimizing peak lung function, and highlights the importance of understanding the underlying mechanisms of PLD during childhood.
Exhaled breath condensate (EBC) is a useful sample type to study in children as it is easily and non-invasively collected.EBC is composed of droplets of epithelial lining fluid (ELF), evolved from all compartments of the lung during tidal breathing.ELF is a complex matrix of compounds, which includes metabolites, and reflects lung tissue biology 12 .Metabolomic methods simultaneously analyse the entire low-molecular weight (< 2000 Da) metabolite content of biological samples and have been applied extensively to EBC in the study of both adult and paediatric respiratory diseases such as COPD, asthma and cystic fibrosis (CF) 13 , showing the ability to discriminate between asthma phenotypes, and evidence of medication altering the EBC metabolome in CF.It is clear metabolomics offers a tool to unravel mechanisms of disease pathogenesis and progression, and identify potential groups of biomarkers in respiratory pathologies.Metabolomic techniques have previously been used on tracheal aspirates obtained during the neonatal period to study the pathogenesis of BPD 14 , finding an increase in metabolites related to hypoxic stress and nitric oxide synthesis.
We hypothesized that the EBC of preterm-born school-aged children with a history of BPD would show altered metabolite content when compared to preterm-born and term-born control children.Therefore, in this exploratory study, we aimed to characterise the metabolome of preterm-born school-aged children with a history of BPD compared to preterm-born and term-born controls.

Participants
This study was conducted on a cohort of children recruited to the Respiratory Health Outcomes in Neonates study (RHiNO, EudraCT: 2015-003712-20) which has been described extensively previously [15][16][17] .In brief, children from a previous study 18 were supplemented with additional preterm-born children identified by the NHS Wales Informatics Service and sent a respiratory and neurodevelopmental questionnaire if they were born ≤ 34 or ≥ 37 weeks' gestation and were aged 7-12 years.Children with significant congenital malformations, cardiopulmonary or neuromuscular disease were excluded.Ethical approval was obtained from the South-West Bristol Research Ethics Committee (15/SW/0289).Parents gave informed written consent and children provided assent.The study was conducted according to the Good Clinical Practice (GCP) guidelines and the Declaration of Helsinki.
Following a home assessment, a subset of responders attended the hospital-based children's research facility for comprehensive clinical examination and respiratory testing including collection of EBC, conducted by a trained nurse and paediatrician between January 2017 and November 2019.Spirometry (MasterScreen Body and PFT systems, Vyaire Medical, Germany) was performed to ATS/ERS guidelines 19 and normalised using Global Lung Initiative (GLI) references 20 .Any respiratory medications were withheld prior to their assessment (short-and long-acting β2-agonists for 8-and 48-h respectively; inhaled corticosteroids for 24 h; and leukotriene receptor antagonists for 48 h) and children were free of respiratory infections for at least three weeks prior to testing.Term-born children who had %FEV 1 > 90% were included as term controls.BPD was defined as oxygen-dependency of 28 days or greater for those born < 32 weeks' gestation and at 56 days of age for those born ≥ 32 weeks' gestation) 21 .Intrauterine growth restriction (IUGR) was defined as birthweight < 10th percentile adjusted for sex and gestation (LMS Growth version 2.77, Medical Research Council, UK).Doctor-diagnosed asthma was self-reported by parents.Neonatal history was corroborated with medical records.Socioeconomic status was assessed using the Welsh Index of Multiple Deprivation 22 scores from 2019, the most contemporaneous available for this cohort.

EBC sampling
EBC was collected in a standardised manner using a cooling tube (RTube®, Respiratory Research Inc., Texas, USA), that was pre-cooled to − 20 °C for at least two hours prior to use, during 10 min of passive tidal breathing, with the participant wearing a nose clip, stopping briefly to swallow saliva if needed, as per manufacturer's instructions.Environmental temperature and humidity remained stable during sampling.The RTube® is a singlepatient, single-use design, preventing cross-contamination, and features a large 'Tee' section to separate saliva from exhaled breath, thereby ensuring collection of ELF and not oropharyngeal secretions.EBC was collected immediately prior to spirometry and, once collected, samples were immediately separated into aliquots and stored at − 80 °C pending analysis.

Metabolome analysis
EBC samples were analysed using Gas Chromatography Time-of-Flight Mass Spectrometry (GCTOF-MS) by the West Coast Metabolomics Centre (University of California, Davis), who have previously published their analytical method 23 .50 μL of each sample was fractionated using an Agilent 6890 gas chromatograph (Agilent, Santa Clara, CA, USA), controlled using Leco ChromaTOF software v2.32 (LECO, St. Joseph, MI, USA), in a Rtx-5Sil MS (Restek, Bellafonte, PA, USA) column (30 m length × 0.25 mm internal diameter with 0.25 μm film made of 95% dimethyl/5%diphenylpolysiloxane).Column temperature was maintained between 50 and 330 °C, with a helium mobile phase.Injection volumes of 0.5 μL were used, with injection temperatures starting at 50 °C, ramped up to a maximum temperature of 250 °C by 12 °C s −1 .Oven temperature program was set to 50 °C for 1 min, then ramped at 20 °C min −1 to 330 °C, and held constant for 5 min.The analytical GC column was protected by a 10 m long empty guard column which was cut by 20 cm intervals whenever the reference mixture QC samples indicated problems caused by column contamination.This sequence of column cuts has previously been validated, with no detrimental effects being detected with respect to peak shapes, absolute or relative metabolite retention times or reproducibility of quantifications.This chromatography method yields excellent retention and separation of primary metabolite classes (amino acids, hydroxyl acids, carbohydrates, sugar acids, sterols, aromatics, nucleosides, amines and miscellaneous compounds) with narrow peak widths of 2-3 s and very good within-series retention time reproducibility of better than 0.2 s absolute deviation of retention times.Automatic liner exchanges after each set of 10 injections were used, which reduces sample carryover for highly lipophilic compounds.

Statistical analysis
Sample demographics were compared using chi-squared or one-way ANOVA with Bonferroni correction tests as appropriate.Metabolite quantities were log 10 transformed and visually inspected for normality.Metabolites with mean and median peak intensities below the mass spectrometer's limit of detection were removed from further analysis to ensure robust statistical comparisons between clinical groups.Fold changes between groups were calculated and log 2 transformed (log 2 FC) for visualization.Independent t-test/ANOVA with post-hoc Bonferroni correction was used to compare metabolite quantities between groups.Metabolite Set Enrichment Analysis (MSEA; identifying biological processes linked to over-represented metabolites) was performed on all metabolites identified with a significantly different quantity between groups using the Small Molecule Pathways Database (SMPDB) 26 , which is based on the Human Metabolome Database (HMDB).Univariable and multivariable linear regression models were used to identify associations between participant characteristics and metabolites of interest identified by MSEA.p < 0.05 was considered statistically significant.All analyses were performed using R v4.0.4 (R Foundation for Statistical Computing, Austria) and MetaboAnalyst v5.0 (www.metab oanal yst.ca) 27 .

Results
From 1426 returned questionnaires and 768 who underwent home assessments, a total of 241 children underwent detailed assessment at the research facility.EBC was successfully collected and analysed from 214 (89%) children with adequate spirometry (Supplementary Fig. 1).Sample demographics are shown in Table 1 21 .Preterm-born children with a history of BPD (BPD) were born at a significantly lower gestational age compared to those without BPD (No BPD) (mean ± SD 27.1 ± 2.1 weeks vs 31.8 ± 1.9, p = < 0.001), with a significantly lower birthweight (1029 ± 415 g vs 1817 ± 493, p = < 0.001).No BPD group was significantly older than the Term group at assessment (10.3 ± 1.1 years vs 9.7 ± 1.1, p = 0.002) but there was no significant age difference between the BPD and the No BPD and Term groups.There was no difference in socioeconomic deprivation scores between the three groups.Percent predicted forced expiratory volume in 1 s (%FEV 1 ) was significantly lower in the BPD group compared to both the No BPD (86.9 ± 15.9 vs 93.2 ± 14.1, p = 0.036) and Term groups (86.9 ± 15.9 vs 104.3 ± 7.1, p = < 0.001).FEV 1 /Forced Vital Capacity (FVC) ratio was also significantly lower in the BPD group compared to the No BPD (0.77 ± 0.10 vs 0.82 ± 0.09, p = 0.002) and Term groups (0.77 ± 0.10 vs 0.85 ± 0.06, p = < 0.001).Percent predicted mid-expiratory flows (%FEF 25-75 ) were also significantly lower in the BPD group compared to No BPD (64.0 ± 25.8 vs 79.3 ± 24.6, p = 0.003) and Term groups (64.0 ± 25.8 vs 94.7 ± 19.1, p = < 0.001).A total of 235 metabolites related to primary metabolism (carbohydrates and sugar phosphates, amino acids, hydroxyl acids, free fatty acids, purines, pyrimidines, aromatics, and exposome-derived chemicals) were successfully detected and identified from the BinBase database, with 128 (54.5%) having mean and median peak intensities greater than the limit of detection, and 38 (16.2%) metabolites being detected above the limit of detection in every sample analysed.Details of all detected metabolites and the number of samples in which they were present are given in Supplementary Table 1.Overall, the metabolite content of EBC was relatively low, with several metabolites close to the limit of detection in multiple samples.
Univariable, unadjusted, linear regression models of the preterm-born cohort studying demographic and lung function characteristics identified that alanine (beta − 0.18, p = 0.025) and urea (− 0.29, 0.013) were only significantly associated with a history of BPD (Table 4).In univariable linear regression models for pyroglutamic acid and ornithine, female sex and history of BPD were both significantly related to a reduced quantity of these metabolites.When combined into a multivariable linear regression model, BPD remained significantly associated with a reduced quantity of pyroglutamic acid (beta − 0.24, p = 0.016) and ornithine (− 0.24, 0.039) (Table 4).No significant associations were noted between these metabolites and current lung function in univariable linear regression models with these metabolites of interest.We observed minimal significantly altered metabolites when comparing the mild and moderate/severe BPD groups (Supplementary Fig. 2).
Figure 3 shows results for ANVOA with post-hoc Bonferroni comparisons between BPD, No BPD and Term groups for alanine, pyroglutamic acid, ornithine and urea.All four metabolites showed a consistent trend of the lowest quantities being present in the BPD group, with both alanine and ornithine having a significantly lower quantity in the BPD group when compared to both the No BPD (p = < 0.001, p = 0.016 respectively) and Term (0.0013, 0.034 respectively) groups.Pyroglutamic acid and urea had a significantly lower quantity in the BPD group compared to No BPD (0.031, 0.0031 respectively), with a near-significant difference when compared to the Term group (0.087, 0.062 respectively).

Discussion
In this exploratory study of the EBC metabolome of preterm-born school-aged children, we have demonstrated significant differences in several metabolites from those with a history of BPD.On comparison to both pretermborn and term-born controls, levels of alanine, pyroglutamic acid, serine, urea and valine were all significantly lower in the BPD group.These five metabolites were detected in > 98% of samples.Alanine and pyroglutamic acid were significantly associated with an alteration of glutathione metabolism.Alanine and urea were significantly associated with an alteration in the urea cycle (with ornithine also being associated when compared to pretermborn controls).Linear regression analyses demonstrated that alanine, pyroglutamic acid, ornithine and urea remained significantly associated with BPD in the preterm-born group when other participant characteristics were also considered.However, linear regression analyses did not show a significant association between any of these metabolites and current lung function parameters.
Pyroglutamic acid, also known as 5-oxoproline, is an intermediary in glutathione synthesis and recycling.Glutathione is a potent antioxidant, and under conditions of oxidative stress, where glutathione is consumed, pyroglutamic acid levels also become low 28 .Alanine concentration also appears to be reduced in metabolomic studies of murine models of pulmonary inflammation, along with pyroglutamic acid 29 .Alanine is a non-essential amino acid that is a constituent of nearly all proteins.Whilst it is not a direct precursor to glutathione, alanine can be converted to pyruvate, a key intermediate of glucose metabolism 30 .Glucose metabolism is an important source of reducing substances, such as NADPH, which are essential in glutathione synthesis 31,32 .Alanine can also be converted to other amino acids, such as serine, which is a precursor to glycine 32 .Glycine is a key, ratelimiting amino acid for glutathione synthesis.Glutathione consumption increases metabolism of glycine, as well as inflammatory conditions reducing glycine availability 31,32 .We noted a significant decrease in serine in our BPD group when compared with the No BPD group, as well as a significant reduction in glycine when compared to the Term group.Taken together, the metabolomic differences observed in the BPD group suggest decreased Glutathione has previously been shown to provide first-line defense against pulmonary oxidative injury.Adult studies have shown that glutathione concentrations in the airway's ELF are many times greater than those seen in plasma 33 , and animal models have shown that pulmonary glutathione depletion enhances oxidant toxicity 34 .In the paediatric population, alterations of glutathione metabolism have been linked with respiratory pathology.A study of ELF in children with severe asthma reported significantly decreased concentration of glutathione, with evidence of glutathione consumption by oxidative stress, further supported by increased levels of hydrogen peroxide (H 2 O 2 ), a powerful oxidant.However, there was no significant association between markers of impaired glutathione metabolism and FEV 1

35
. Impaired glutathione metabolism has also been associated with impaired macrophage function in the airways of children with severe asthma, with glutathione supplementation restoring macrophage function 36 .Reduced concentration of glutathione secondary to oxidative stress has also been described in bronchoalveolar lavage fluid (BAL) obtained from children with cystic fibrosis, with those experiencing an infective exacerbation having a further decreased concentration 37 .
Previous mechanistic studies of BPD in later childhood and adulthood have implicated cytotoxic CD8 + T-lymphoytes 38 , elevated neutrophils and pro-inflammatory cytokines 39 , and thickened basement membranes and airway remodeling 40 .However, to our knowledge, this is the first study to analyze the EBC metabolome of preterm-born children with a history of BPD.Previous studies in preterm infants have linked increases in pulmonary oxidative stress and glutathione metabolism to the development of BPD.In a small study of BAL from preterm infants born < 34 weeks' gestation, lower glutathione levels on the first day of life were associated with increased development of BPD at 36 weeks' gestation 41 .This study was performed before the routine use of surfactant replacement therapy.A further study in the post-surfactant era of infants born at < 32 weeks' gestation reported lower BAL glutathione levels in the first 24 h of life in those who later developed BPD, lower glutathione levels in those who had delayed surfactant administration, and higher concentrations of malondialdehyde, suggestive of oxidative damage, in the BPD group 42 .Animal models have further supported the role of glutathione in lung injury, with glutathione deficient mice having impaired tolerance of oxidative stress, and abnormal early lung development 43 .Whilst glutathione metabolism has been implicated in our study by MSEA, we did not detect glutathione in either its reduced or oxidized form.However, glutathione has a short half-life of approximately ten minutes, which can make it's detection challenging 44 .One previous study has examined oxidative stress in the airways of preterm-born adolescents, measuring 8-isoprostane (a product of lipid peroxidation in the presence of oxygen free radicals) in EBC, both with and without BPD.These individuals, born during the peri-surfactant era, demonstrated evidence of persistent airway oxidative stress, with increased 8-isoprostane when compared to term-born controls 45 .However, in this study no significant difference was observed for 8-isoprostane between BPD and No BPD groups, and no correlation was seen between 8-isoprostane levels and spirometry values.
Previous metabolomic studies of BPD, with similar analytical techniques to our study, have focused on preterm-born infants in the neonatal period.A study of tracheal aspirates taken from infants born < 30 weeks' gestation using metabolomic techniques reported that 19 metabolites discriminated infants who subsequently did or did not develop BPD 14 , including alterations in amino acids (citrulline and symmetric dimehtlyarginine) involved in nitric oxide metabolism, as well as an increase in serine.The authors also observed increased acylcarnitines which are released after β-oxidation of fatty acids during oxidative stress.In contrast, another metabolomic study of tracheal aspirates from infants ≤ 28 weeks' gestation noted that early decreases in fattyacid metabolism, particularly the fatty acid β-oxidation pathway, may predispose infants to developing BPD 46 .A nuclear magnetic resonance metabolomics study of urine from 18 infants born < 28 weeks' gestation showed decreased glycine levels in those who subsequently developed BPD, similarly to our study, also suggesting impaired glutathione metabolism.The authors also found increased alanine in the BPD group, which they attributed to increased cellular metabolism demands, due to an inflammatory process 47 .These authors studied infants in the first week of life, where the pathophysiology is respiratory distress syndrome and pulmonary surfactant deficiency, as opposed to the chronic inflammation seen in BPD, potentially explaining the different alanine levels we observed in our study.
In our study, we observed significant decreases of alanine, ornithine, and urea in the BPD group, with MSEA linking these changes to a significant decrease in urea cycle metabolism.The urea cycle removes ammonia, produced during protein catabolism, preventing cellular toxicity.Animal models have demonstrated increased ammonia levels lead to intracellular production of reactive oxygen species, induction of cellular apoptosis in bovine epithelial cells, increased inflammatory cytokines and repression of DNA repair-related genes in porcine Type II alveolar epithelial cells 48,49 .We also observed a significant reduction in histidine in our BPD group when compared to both the No BPD and Term groups.Histidine, an essential amino acid, is metabolised to histamine and can affect the contractility of bronchial smooth muscle and cause airway oedema 50 .Histidine itself has also been reported to have antioxidant properties, being a scavenger of free hydroxyl and singlet oxygen radicals  is thought to play a role in childhood asthma pathogenesis, with a consequent increase in proline production leading to collagen deposition 52 .Whilst elevated FE NO is used clinically as a biomarker of asthma 53 , methylated products of arginine can inhibit NOS activity and contribute to airway oxidative stress in specific phenotypes of asthma associated with obesity 54 .The reduced quantities of metabolites linked to protection from reactive oxidant species in those with BPD implied by our results suggest a persistent deficit since the neonatal period, especially for glutathione, however, this is speculative.We did not observe any associations between metabolic processes involved with oxidative stress and current lung function.This may be due to other processes such as functional and structural abnormalities also significantly contributing to the development of PLD as we have recently reported 55,56 .However, one previous study using superoxide dismutase did not decrease rates of BPD in the neonatal period but was associated with decreased respiratory symptoms at one year of age 57 .Whether such treatments are beneficial for PLD will require further study.In addition, longitudinal metabolome analysis may reveal emerging mechanisms related to lung function decline.This study represents one of the largest metabolomic studies of a clinical cohort.The significantly altered metabolites of interest in the BPD group were detected in all or nearly all samples analyzed, ensuring robust results.Using an untargeted metabolomics approach, we have been able to identify patterns of changes in multiple metabolites which we have been able to link with biological processes.We have studied a cohort of children who had experienced contemporary standard of neonatal care, and by using EBC we have been able to directly sample ELF, which is representative of airway biochemistry in a simple and well-tolerated manner.Although EBC volumes varied between our clinical groups, linear regression analyses did not reveal a significant association between metabolite quantities and EBC volume.As the overall metabolite content of EBC was low, as has been previously reported 58 , there may have been metabolites present that were below the limit of detection for the mass spectrometry method used.We combined our mild and moderate/severe BPD groups as few differences were noted for metabolites between these two groups thus it was reasonable to combine these two groups.Dietary intake has recently been shown to modulate the breath metabolome 59 , however we had insufficient nutritional data to adjust for this potential confounder.Similarly, whilst data was initially collected on antenatal and household smoking, overall rates in the preterm-born cohort were low suggesting high recall bias, and therefore were not included in our analyses.Whilst a reference metabolite would be useful to normalize metabolite concentrations, as in urine metabolomics 60 , this is currently not possible with EBC samples 61 .Ideally, our findings should be replicated using a validation cohort, however we are limited by the number of large contemporaneous cohorts available to study.
In conclusion, this exploratory mass spectrometry-based analysis of the EBC metabolome has revealed significant reductions in metabolites related to antioxidant pathways in the airways of preterm-born schoolaged children with a history of BPD, many years after the initial pulmonary insult.Future work should focus on therapeutic strategies to improve antioxidant mechanisms in these children, with a possible improvement in longer-term respiratory outcomes.

Figure 1 .
Figure 1.Volcano plots demonstrating significantly altered metabolites between groups (A) BPD vs No BPD (B) BPD vs Term.Vertical line represents a Log 2 FC of 0. Horizontal line is equivalent to p-value 0.05.Size of point is relative to number of samples in which metabolite was detected.Metabolite name given if p < 0.05.BPD, Bronchopulmonary dysplasia; Log 2 FC, log 2 fold-change between groups.

Figure 2 .
Figure 2. Graphic representation of glutathione metabolism and the urea cycle, highlighting metabolites detected in this analysis and those with a significantly reduced concentration in the BPD group.LOOH, lipid hydroperoxide; LOH, lipid hydroxide; ROS, reactive oxygen species; NADP, nicotinamide adenine dinucleotide phosphate; NADPH, nicotinamide adenine dinucleotide phosphate hydrogen; NADH, nicotinamide adenine dinucleotide + hydrogen.

Figure 3 .
Figure 3. Violin Plots of Significantly Altered Metabolites in BPD group.Black dot and bars give mean and standard error of the mean (SEM).Horizontal bars give p-values from ANOVA with post-hoc Bonferroni correction for between group comparisons.

Table 2 .
Significantly different metabolites between phenotypes.BPD, Bronchopulmonary dysplasia; m/z, mass-to-charge ratio; Log 2 FC, Log 2 fold change.p values represent between group comparisons using t-test.

Table 3 .
Metabolite set enrichment analysis demonstrating altered biological processes implicated by significantly altered metabolite quantities.

Table 4 .
Linear regression models for preterm-born cohort.Univariable regression models where p < 0.1 included in multivariable model.Multivariable models not created where only one variable has a p < 0.1 in univariable models.IUGR, intrauterine growth restriction; BPD, Bronchopulmonary Dysplasia; BMI, body mass index; FEV 1 , forced expiratory volume in 1 s; FVC, forced vital capacity; EBC, exhaled breath condensate.* and [bold] denotes p < 0.05.
51d inhibiting fatty-acid oxidation during in vitro studies51.Lower quantities of urea and ornithine in the BPD group were also significantly linked to arginine and proline metabolism.Arginine is metabolized into either nitric oxide by nitric oxide synthetase (NOS) or into urea and ornithine by arginase.Increased arginase activity